This disclosure relates to methods for polymerization of nucleic acids (or nucleotides) using activation by polyphosphorolysis (APP) reactions. Polymerization may be carried out using primers having 3′-ends blocked with dideoxynucleotides in a reaction mixture containing a balance of pyrophosphate (PPi) and dNTP. These methods have been previously described in, for example, U.S. Pat. Nos. 6,534,269 B2, 7,033,763 B2, 7,238,480, 7,105,298, 7,504,221, and 7,919,253. Similar methods are also described in U.S. Pat. No. 7,745,125. These methods have many applications, including detection of rare alleles in the presence of wild type alleles (e.g., one mutant allele in the presence of 106-109 wild type alleles). The methods are useful to, for example, detect minimal residual disease-causing agent(s) (e.g., rare remaining cancer cells during remission, especially mutations in the p53 gene or other tumor suppressor genes previously identified within the tumors), and/or measure mutation load (e.g., the frequency of specific somatic mutations present in normal tissues, such as blood or urine). Other applications of such methods are known to those of skill in the art.
Those of skill in the art desire improved methods for carrying out such methods. It has been surprisingly found that several other compounds are also capable of deblocking of nucleic acids. Described herein are methods for performing an APP reaction using various polyphosphorolyzing agents. Advantages of these methods will be apparent from the following description of such improved methods.